1. Field of the Invention
The present invention relates to a method for activating a denatured protein. In particular, the present invention relates to a method for activating a denatured protein which includes the steps of: adding a detergent to a denatured protein to form a protein-detergent complex; and adding high-molecular weight amylose to the protein-detergent complex so that the high-molecular weight amylose removes the detergent. Moreover, the present invention relates to a protein refolding kit including at least one kind of high-molecular weight amylose and at least one kind of detergent.
2. Description of the Related Art
A protein includes a polypeptide chain consisting of a plurality of L-xcex1-amino acids which are linked via peptide bonds. The order in a sequence of amino acids of a protein is referred to as a primary structure. An actual protein assumes a three-dimensional conformation referred to as a xe2x80x9chigher-order structurexe2x80x9d. In order for a protein to function, it is essential that the protein assumes a proper higher-order structure. Higher-order structures of proteins include secondary structures, tertiary structures, and quaternary structures. Examples of secondary structures include the xcex1 helix structure, xcex2 sheet structure, and the like. Such secondary structures may be further folded to give tertiary structures. Examples of bonds which make for the stabilization of tertiary structures include hydrophobic bonds, hydrogen bonds, and Sxe2x80x94S bonds between cysteine residues. Through the non-covalent association of a plurality of polypeptide chains each assuming a tertiary structure, a quaternary structure results.
A structure which is inherently assumed by a protein molecule under substantially physiological conditions is referred to as a xe2x80x9cnativexe2x80x9d structure. The term xe2x80x9cdenaturationxe2x80x9d refers to the alteration of the physicochemincal properties of a protein from its native state, which may be induced by various factors which cause the destruction of a higher-order structure without causing changes in the primary structure. The destruction of a higher-order structure of a protein may occur due to physical causes, e.g., heating, freezing, irradiation by ultraviolet or X rays, as well as chemical causes, e.g., extremely alkaline conditions, denaturing agents such as organic solvents, urea, and guanidine hydrochloride, or detergents. As a result of denaturation, the structure of a protein may change from a compact structure with orderly folds to an irregular aggregated structure, or a random structure in which the folds have been unfolded. As used herein, a xe2x80x9cdenatured statexe2x80x9d includes both irregularly aggregated states and unfolded states.
A so-called xe2x80x9crefolding techniquexe2x80x9d, i.e., a technique for refolding a protein which is in a denatured state as defined above in such a manner as to restore a proper higher-order structure, has very important implications in the industrial utilization of proteins. For example, an enzyme which has been utilized for a certain reaction for producing a substance may be denatured (through heating or the addition of a denaturing agent), whereby its enzyme activity is lost so that the reaction stops; after the resultant product has been recovered, the denatured enzyme may be refolded so as to restore a proper higher-order structure, i.e., activated. Thus, such an enzyme can be put to further reuse.
When heterogeneous proteins are produced in an E. coli host, a number of proteins may often become insoluble and inactive substances, referred to as xe2x80x9cinclusion bodiesxe2x80x9d. However, with a refolding ability, such an insoluble protein may be first solubilized by a denaturing agent such as urea or guanidine hydrochloride, and thereafter refolded so as to restore a proper higher-order structure, i.e., activated.
The technique of refolding a denatured protein for restoring a proper higher-order structure is associated with two specific problems to be solved: the first problem is how to prevent aggregation of protein; the second problem is how to properly refold the unfolded protein molecules back into a protein.
In the in-vivo folding of a protein, a class of assisting proteins, termed xe2x80x9cmolecular chaperonsxe2x80x9d, are known to be involved in the above two steps. Molecular chaperons are proteins which bind to a protein which has just been synthesized so that the protein is prevented from being folded in an irregular manner and can be readily transported, and/or assist in the folding process of proteins which would otherwise have difficulties in folding.
A xe2x80x9cnascent proteinxe2x80x9d is a protein which has just been translated in vivo and has not assumed a proper higher-order structure. Immediately after the completion of translation, a nascent protein is bound by a class of molecular chaperons called DnaJ, etc. These molecular chaperons operate upstream of the folding process so as to prevent nascent protein from aggregating or assuming abnormal structures. Thereafter, another molecular chaperon called GroE, which operates downstream of the folding process, acts on the nascent protein. Owing to the action of GroE, the nascent protein gradually begins to assume a proper higher-order structure, until it is finally folded, into an active protein. In the course of the folding process, the molecular chaperons which assisted in the folding leave the nascent protein.
In recent years, several attempts have been made to construct artificial chaperons with a view to reproducing the in-vivo functions of molecular chaperons In vitro (e.g., in a test tube) and restoring the activity of a denatured protein in vitro. Daugherty et al. (J. Biol. Chem., 273,3961-33971 (1998)) reported a method of refolding a denatured protein by using artificial chaperons. The reported method employs a non-ionic detergent designated Triton X-100, and a polyoxyethylene-type detergent having a short alkyl group chain, as artificial chaperons which function to prevent aggregation of proteins. After a protein-detergent complex is formed by using these artificial chaperons, xcex2 cyclodextrin (hereinafter abbreviated as xe2x80x9cxcex2 CDxe2x80x9d), which is a low-molecular weight cyclic xcex1-1,4-glucan, is added as a substance (hereinafter also referred to as a xe2x80x9cdetergent removing agentxe2x80x9d) for causing removal of the detergents. Thus, the detergents are gradually removed from the protein-detergent complex, thereby allowing the denatured protein to naturally assume a higher-order structure.
Thereafter, Silvakama Sundari et. al (FEBS Lett., 443, 215-219 (1999) ) discloses that, not only a low-molecular weight cyclic xcex1-1,4-glucan (cyclodextrin), but also a low-molecular weight straight-chain xcex1-1,4-glucan can be effective as a substance for causing gradual removal of detergents from a protein-detergent complex in a similar artificial chaperon system.
More recently, Machida et al. (Japanese Patent Application No. 2000-71533) is a study specifically into the effects of various combinations of detergents and detergent removing agents on the restoration of the activity of three different proteins in a similar artificial chaperon system, reporting the following results:
(1) High-molecular weight cyclic xcex1-1,4-glucan (having a polymerization degree of about 40 to about 150) enables faster and more effective restoration of protein activity than low-molecular weight cyclic xcex1-1,4-glucan (having a polymerization degree of about 6 to about 8).
(2) Each protein may have its activity restored to various degrees depending on the detergents and the detergent removing agents used. Therefore, for a higher level restoration of activity, it is essential to select appropriate combinations of detergents and detergent removing agents in accordance with the protein to be restored.
Thus, it has been indicated that the artificial chaperon technology is very effective for the activation of denatured proteins. On the other hand, it has also been learned that the degree of activity restoration of a denatured protein may substantially vary depending on the detergents and the detergent removing agents used.
A large number of detergents are known in the art to be usable as artificial chaperons.
As for detergent removing agents, high-molecular weight and low-molecular weight cyclic xcex1-1,4-glucans (Japanese Patent Application No. 2000-71533, supra) and low-molecular weight straight-chain xcex1-1,4-glucan (Silvakama Sundari et al., supra) are currently known. These glucans are found to be every effective for the activation of denatured proteins.
Amyloses are commercially available, such as: low-molecular weight amylose (average molecular weights: about 2900), which is obtained by decomposing starch with a debranching enzyme such as isoamylase; and amylose fractions after removing amylopectin from natural starch. Low-molecular weight amylose is not preferably used for the activation of denatured proteins because of its small molecular weight and poor inclusion ability. Amylose fractions are not preferably used for the activation of denatured proteins because of their extremely poor water solubility, high impurity contents (e.g., fat), branch structures associated with xcex1-1,6-bonds, and broad molecular weight distribution.
On the other hand, a similar glucose polymers, xcex1-1,6-glucan, has been shown to have no ability to activate denatured proteins (Silvakama Sundari et al., supra). It has also been indicated that the same cyclic xcex1-1,4-glucan may have different degrees of protein activation action depending on the polymerization degree thereof. That is, a cyclic xcex1-1,4-glucan having a high polymerization degree provides a higher degree of protein activation action than a cyclic xcex1-1,4-glucan having a low polymerization degree (Japanese Patent Application No. 2000-71533, supra).
Thus, it is already known that polymers of glucose may have different degrees of protein activation action depending on the mode of bonding, polymerization degree, and the like. Thus, as for detergent removing agents, there is no adequate understanding as to which substances can be expected to have good effects. Hence, it is difficult for those skilled in the art to select proper detergent removing agents.
While the hitherto undertaken studies find the highest degree of protein activation action in cyclic xcex1-1,4-glucan having a high polymerization degree, such a cyclic xcex1-1,4-glucan having a high polymerization degree is difficult to produce in practice, and has not been trade commercially available yet. Thus, there are some practicality concerns.
Against such a back ground, there is a need for developing a detergent removing agent which has a high activation action on denatured proteins and which has a high practicality. Moreover, there is a need for a useful and highly practical method for activating a denatured protein, where the protein may have e poor spontaneous folding ability and have difficulties in assuming a proper higher-order structure, or even cannot assume a higher-order structure without the assistance of molecular chaperons, such that the protein can be refolded in a relatively short time so as to assume a proper higher-order structure for acquiring activity.
According to one aspect of the present invention, there is provided a method for activating a denatured protein, including the steps of: adding a detergent to the denatured protein to allow a protein-detergent complex to be formed; and adding high-molecular weight amylose to the protein-detergent complex so that the high-molecular weight amylose removes the detergent.
In one embodiment of the invention, the method further includes the step of adding a denaturing agent to the denatured protein to cause the denatured protein to be in an unfolded state, wherein the step of adding the denaturing agent is performed before the step of adding the detergent to the denatured protein.
In another embodiment of the invention, the high-molecular weight amylose is an inclusion compound which includes a lower alcohol.
In still another embodiment of the invention, the lower alcohol is butanol.
In still another embodiment of the invention, the high-molecular weight amylose has an average molecular weight in the range of about 10,000 to about 20,000,000.
In still another embodiment of the invention, the high-molecular weight amylose has an average molecular weight in the rage of about 20,000 to about 11,000,000.
In still another embodiment of the invention, the high-molecular weight amylose is an amylose which is synthesized by using an enzyme.
In still another embodiment of the invention, the detergent is selected from the group consisting of: polyoxyethylene sorbitan ester, polyoxyethylene dodecyl ether, polyoxyethylene fatty acid ester, sucrose fatty acid ester, cetyltrimethylammonium bromide, sodium deoxycholate, hexadecyltrimethylammonium bromide, and myristylsulfobetaine.
In still another embodiment of the invention, the denatured protein includes an xcex1 helix structural region.
In still another embodiment of the invention, the denatured protein includes a xcex2 sheet structural region.
In still another embodiment of the invention, the denatured protein includes an intramolecular Sxe2x80x94S bond.
According to another aspect of the present invention, there is provided a method for activating a denatured protein including an xcex1 helix structural region, including the steps of: adding an excess of a polyoxyethylene type detergent complex to be formed, thereby preventing aggregation of the protein; and adding high-molecular weight amylose to the protein-detergent complex so that the high-molecular weight amylose removes the detergent, thereby allowing the protein to be folded again into a higher-order structure for acquiring activity, thus activating the protein.
In one embodiment of the invention, the polyoxyethylene type detergent is selected from the group consisting of: polyoxyethylene sorbitan ester, polyoxyethylene dodecyl ether,polyoxyethylene fatty acid ester, and sucrose fatty acid ester.
According to yet another aspect of the present invention, there is provided a method for activating a denatured protein including a xcex2 sheet structural region, including the steps of: adding an excess of an ionic type detergent to the denatured protein to allow a protein-detergent complex to be formed, thereby preventing aggregation of the protein; and adding high-molecular weight amylose to the protein-detergent complex so that the high-molecular weight amylose removes the detergent thereby allowing the protein to be folded again into a higher-order structure for acquiring activity, thus activating the protein.
According to still another aspect of the present invention, there is provided a method for activating a denatured protein including an intramolecular Sxe2x80x94S bond, including the steps of: adding an excess of an ionic type detergent to the denatured protein to allow a protein-detergent complex to be formed, thereby preventing aggregation of the protein; and adding high-molecular weight amylose to the protein-detergent complex so that the high-molecular weight amylose removes the detergent, thereby allowing the protein to be folded again into a higher-order structure for acquiring activity, thus activating the protein.
In one embodiment of the invention, the ionic type detergent is selected from the group consisting of: cetyltrimethylammonium bromide, sodium deoxycholate, hexadecyltrimethylammonium bromide, and myristylsulfobetaine.
In another embodiment of the invention, the ionic type detergent is selected from the group consisting of: cetyltrimethylammonium bromide, sodium deoxycholate, hexadecyltrimethylammonium bromide, and myristylsulfobetaine.
According to still another aspect of the present invention, there is provided a kit for refolding a protein, including at least one kit of high-molecular weight amylose and at least one kit of detergent.
In one embodiment of the invention, the high-molecular weight amylose is an inclusion compound which includes a lower alcohol.
In another embodiment of the invention, the lower alcohol is butanol.
In still another embodiment of the invention, the high-molecular weight amylose has an average molecular weight in the range of about 10,000 to about 20,000,000.
In still another embodiment of the invention, the high-molecular weight amylose has an average molecular weight in the range of about 20,000 to about 11,000,000.
In still another embodiment of the invention, the high-molecular weight amylose is an amylose which is synthesized by using an enzyme.
In still another embodiment of the invention, the detergent is selected from the group consisting of: polyoxyethylene sorbitan ester, polyoxyethylene dodecyl ether, polyoxyethylene fatty acid ester, sucrose fatty acid ester, cetyltrimethylammonium bromide, sodium deoxycholate, hexadecyltrimethylammonium bromide, and myristylsulfobetaine.
After much research effort, the inventors have found that a high-molecular weight amylose, which is a straight-chain xcex1-1,4-glucan having a high polymerization degree, functions, more effectively as a detergent removing agent than a straight-chain xcex1-1,4-glucan having a low molecular weight, and therefore is useful for the efficient activation of a denatured protein. The Inventors further found that a method for activating a denatured protein which employs a high-molecular weight amylose can be generically utilized for various denatured proteins, thereby accomplishing the present invention. High-molecular weight amylose is more practical than high-molecular weight cyclic xcex1-1,4-glucan because high-molecular weight amylose can be supplied in large quantities through synthesis employing phosphorylase.
By combining high-molecular weight amylose and various detergents, a protein refolding kit having a high practicality and generic applicability can be provided according to the present invention.
Thus, the invention described herein makes possible the advantages of (1) providing a method for efficiently activating a denatured protein; and (2) providing a protein refolding kit.
These and other advantages of the present invention will become apparent to those skilled in the art upon reading and understanding the following detailed description with reference to the accompanying figures.